Male C57BL/6 mice aged 8–12 weeks were purchased from the National Experimental Animal Center (Taipei, Taiwan) and housed at the Laboratory Animal Center of Taipei Veterans General Hospital (Taipei, Taiwan). They were kept under a 12/12-h light/dark cycle and had access to food and water ad libitum. All experimental procedures followed committee-approved protocols for institutional animal care and use (Taipei Veterans General Hospital IACUC no. 2020-258).
Endotoxin-induced lung injury in mice is an experimental animal model of ALI. Here, we used a model of endotoxin-induced ALI established in our previous work [8,9,10,11,12,13]. Briefly, to induce ALI, anesthetized mice received an intratracheal instillation of LPS from Escherichia coli (0111:B4; Sigma-Aldrich, St. Louis, MO) at a dose of 5 mg/kg in 50 µL PBS. Control mice received intratracheal instillations of 50 µL PBS daily for 5 days. In the LPS group, mice received 50 µL PBS daily for four days (Days 1–4) and were treated with ALI-inducing LPS on Day 5.
E-cigarette with 60%VG and 40%PG is widely used. Additional file 1: Fig. S1 revealed that lung injury induced by 30% VG solution was equal to lung injury induced by 60%VG solution. The toxicity of 30% VG solution has been confirmed in a previous study using human airway cell lines . Intratracheal instillations of the 20% mannitol (1098 mOsm/L) did not induce lung injury and fibrosis in mice (Additional file 1: Fig. S1).
Intratracheal instillations of 30% VG solution was used to induce VG-induced ALI. In the VG group, mice received a 50 µL 30% VG solution daily on Days 1–4 and received PBS on Day 5. In the VG + LPS group, mice received a 50 µL 30% VG solution daily on Days 1–4 and received LPS on Day 5. In the LPS + p38 inhibitor and VG + LPS + p38 inhibitor groups, each mouse received an intraperitoneal injection of 5 mg/kg p38 inhibitor (SB203580, #tlrl-sb20, InvivoGen, San Diego, CA) 1 h before LPS instillation . In the VG + p38 inhibitor group, each mouse received an intraperitoneal injection of 5 mg/kg p38 inhibitor, 1 h before VG instillation on Day 1. The p38 inhibitor was dissolved in 30 µL dimethyl sulfoxide and mixed with 270 µL normal saline.
Mice were euthanized 24 h after the last intratracheal injection. Lung tissues were collected from each mouse to assess ALI via H&E histology, IHC, IF, and western blot analyses.
Histological and IHC analyses
Lung tissue was excised 24 h after LPS-induced lung injury, fixed in 4% paraformaldehyde for 10 min, embedded in paraffin, and cut into 4-µm-thick sections. Staining for Ly6G (LS-C36561, 1:100; LifeSpan Biosciences, Seattle, WA), MPO (SC-52707, 1:100, Santa Cruz Biotechnology, Dallas, TX), VLA-4 (#8440S, 1:1000; Cell Signaling, Danvers, MA), VCAM-1 (#14694, 1:1000; Cell Signaling), and TGF-β (ab66043, 1:100, Abcam, Cambridge, UK) was performed using Envision® + Dual Link System-HRP (DAB+) kits (K4065; DAKO, Carpinteria, CA). The sections were deparaffinized in xylene, dehydrated in ethanol, and then heated in 0.01 M citrate buffer (pH 6.0). Endogenous peroxidase activity was inactivated in 3% H2O2 for 10 min at room temperature (RT), and the sections were blocked with blocking buffer. Secondary anti-rabbit antibody-coated polymer peroxidase complexes were applied for 30 min at RT, followed by substrate/chromogen incubation for 5–15 s at RT. The sections were counterstained with hematoxylin (109249; Merck) for 10 s and then washed in running water for 10 min. They were observed and photographed with an Olympus AX80 fluorescence microscope (Olympus America, Melville, NY). The percentage of IHC signal per photographed field was determined with Image-Pro Plus software (Media Cybernetics, Inc., Silver Spring, MD).
Lung injury scoring
Two investigators evaluated each H&E-stained slide independently while blind to the groups. To generate the lung injury score as an index of ALI severity, 300 alveoli were counted on each slide at 400× magnification. Within each field, points were assigned according to established criteria [8,9,10,11,12,13]. We calculated the scores using the following formula: Lung injury score = [(alveolar hemorrhage points/no. of fields) + 2 × (alveolar infiltrate points/no. of fields) + 3 × (fibrin points/no. of fields) + (alveolar septal congestion/no. of fields)]/total number of alveoli counted.
Masson’s trichrome staining
Lung specimens were fixed in 4% paraformaldehyde for 10 min, embedded in paraffin, and cut into 3-µm-thick sections. The sections were stained with a Trichrome Stain Kit (#ab150686, Abcam, Cambridge, UK) according to the manufacturer’s instructions.
Two investigators evaluated each Masson’s trichrome-stained slide independently and blind to group assignments. Points were assigned within each field pursuant to the predetermined criteria used in a previous study .
Mouse lung tissues were homogenized in lysis buffer [RIPA lysis buffer (475 uL), Halt protease inhibitor cocktail (5 uL), and 0.1 M Na3VO4 (20 uL); Thermo Fisher Scientific, MA], centrifuged at 20,000 rpm at 4 °C for 10 min, and stored at − 20 °C. Equal amounts of protein homogenate were resolved on 7.5–10% sodium dodecyl sulphate–polyacrylamide electrophoresis gels and transferred to polyvinylidene fluoride membranes. The blots were blocked in Tris-buffered saline with Tween (TBST) containing 5% milk and probed with primary antibodies to VLA-4 (#8440S, 1:1000; Cell Signaling), VCAM-1 (#14694, 1:1000; Cell Signaling), p38 (#9212S, 1:1000; Cell Signaling), phosphorylated (p)-p38 (#9211, 1:1000; Cell Signaling), collagen-1 (ab34710, 1:1000; Abcam), TGF-β (ab66043, 1:1000, Abcam), and β-actin (20536-1-A, 1:5000; Proteintech). The blots were washed in TBST, incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit immunoglobulin G; H&L, ab6721; Abcam], and detected using an enhanced chemiluminescence substrate (Pierce Biochemicals). Each blot was exposed to film, and the densitometry of the immunoreactive bands was performed in ImageJ.
Cells from BALF and blood were subjected to cytospinning, fixed, permeabilized, and stained with Ly6G (LS-C36561, 1:100; LifeSpan Biosciences) or MPO (ab9525, 1:100, abcam) antibodies as primary antibodies. The following day, goat anti-rabbit IgG (H&L) Alexa Fluor® 488 (1:400, ab150077; Abcam) or goat anti-rabbit IgG (H&L) Cy5® (ab6564, 1:400; Abcam) was incubated as a secondary antibody at 37 °C for 2 h. Cell nuclei were counterstained with DAPI (H-1200; Vector Laboratories, CA). Images of the cells were obtained under a Fluoview confocal microscope (FV10i; Olympus).
To limit the variability of each experimental condition, all mice were prepared and studied at the same time. Separate groups of mice were used for lung injury scoring, IHC, flow cytometry, and migration assays. The data are presented as means ± standard errors or means ± standard deviations and were analyzed using a one-way analysis of variance and the Tukey–Kramer multiple comparisons test (for multiple groups) or Student’s t Test (for two groups). P values < 0.05 were considered statistically significant.